The present invention provides methods for the construction of custom microarrays using information from public databases. One preferred embodiment provides the novel cDNA array, the ImmunoChip. The ImmunoChip is useful for diagnostic procedures and screening involving immunological-based sample materials. In preferred embodiments, the ImmunoChip is used in experiments and screens in innate and adaptive basic immunology, tumor immunology, cancer biology of immune cells, aging, drug testing, infection immunology, autoimmune diseases, arthritis, allergy, and vaccine development against these diseases.
The purpose of the microarray technology is to associate gene functions with sequences obtained by genome or expressed sequence tags (EST) projects (e.g., the Human Genome Project). Since the genome projects for most model organisms are not yet completed, EST databases are presently the ultimate source for construction of custom microarrays. Currently, more than 6.4 million different ESTs from cDNA libraries representing diverse cells and tissues for various model organisms are publicly available. Bioinformatics algorithms such as Unigene, assemble these ESTs into clusters that tentatively define distinct genes. However, despite these rich resources, a method is missing that would allow for the selection of relevant clusters and to identify the biological best representative EST clone within these clusters for the construction of a microarray. In other words, current methods for the selection of DNA clones for incorporation into an array do not allow for the efficient selection of clones that are specific for cell type, tissue type, etc., without undue redundancy What is needed is a method for the efficient construction of microarrays that are specific for cell types, tissues, organs and diseases (e.g., cancer) without redundancy.
The present invention provides methods for the selection of DNA sequences for incorporation into DNA arrays, in which the methods comprises novel algorithms. The methods of the present invention facilitate the construction of DNA microarrays that are non-redundant and specific for the desired tissue, organ, species, developmental stage or disease. By using this method, the xe2x80x9cImmunoChipxe2x80x9d was constructed. The construction of this immunologically relevant microarray integrates (non-redundantly) the expression information of ESTs from known and unknown genes from all immune cells and lymphoid organs in the developing and adult organism.
The present invention contemplates the utilization of sequences selected by the methods of the present invention specific for any tissue type, cell type, organ and/or disease. Indeed, the present invention is not limited to any specific type of tissue, cell or disease. For example, tissue types that find use with the present invention include, but are not limited to lung, heart, muscle, liver, skin, brain, testicle, thymus, kidney, spleen, breast, etc., or a combination of thereof. Thus, many types of tissue will find use with the present invention. In a preferred embodiment, immune tissue (i.e., tissue that is involved in the immune response in some manner) is contemplated. In addition, the present invention is not limited to any particular type of cell. Indeed, many cell types are contemplated. For example, cells including, but not limited to blood cells, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, fibroblastic cells, chondrocytes, epithelial cells, cells of the reticuloendothelial system, etc., will find use in the present invention. In a preferred embodiment, immune cells are utilized. In addition, the present invention is not limited to any specific or particular type of disease. Indeed, many types of disease are contemplated. In a preferred embodiment, the disease is cancer. However, the present invention is not limited to any specific types of cancer, as many types of cancer are contemplated. For example, cancers such as leukemia, as well as those of the lung, stomach, skin, brain, liver, prostate, testes, bone marrow, bone, breast, intestinal, etc., will find use with the present invention. In other embodiments, other diseases are contemplated. Furthermore, in some embodiments, the present invention finds use in monitoring the progression of a particular disease by assessing changes in the detection and/or levels of various cell markers associated with the disease.
The present invention also provides methods and compositions for screening drug treatments for diseases. The present invention is not limited to any particular drug treatment. Drugs identified using the methods of the present invention include newly recognized compounds, as well as compounds that have already been established for treatment (e.g., compounds used for other purposes), as well as experimental treatments. In alternative embodiments, the present invention provides methods and compositions to monitor the effect of a particular drug or drugs by studying the change in detection of various cell markers following administration of the drug(s) to a subject or in cell culture.
The present invention also provides compositions and methods for the construction of non-redundant DNA microarrays using a bioinformatics approach. Additionally, the present invention provides means for the selection of DNA sequences from public and private databases. However, the present invention is not limited to any particular databases, as many public and private databases find use with the present invention. For example, databases, including, but not limited to gene databases (e.g., GenBank), image clone databases, bacterial clone databases, viral databases, EST databases and phage databases find use with the present invention. Additionally, the present invention provides means for the selection of DNA sequences from the literature.
The present invention provides methods and compositions for the construction of an ImmunoChip for any species of animal having an immune system. Thus, it is not intended that the ImmunoChip be limited to any particular species of animal having an immune system. For example, the present invention contemplates the production of ImmunoChips from various species (e.g., bovines, ovines, lagomorphs, caprines, porcines, primates, including humans, canines, felines, rodents [e.g., mice and rats], equines, avians, reptiles, etc.). In a preferred embodiment, the organism is a murine species.
In addition, the present invention provides methods and compositions for the construction of a DNA array for any species of organism. Indeed, the present invention is not limited to any particular species. For example, the species include plants, animals, bacteria, viruses, and any other organisms. However, in a preferred embodiment, the species is an animal species, and in a particularly preferred embodiment, the species is a murine species.
The present invention provides bioinformatics approaches using public and private databases to generate specific and customizable microarrays. In a preferred embodiment, the array is a unique cDNA microarray specifically designed for immunology research. In a particularly preferred embodiment, a microarray of the present invention was constructed in two steps. First, immunological relevant clusters were selected using literature and expression information. Second, the best representative clones for each of the selected clusters were identified. The array comprised representative clones for more than 13,389 different immunological clusters (See, Table 1, attached hereto), or a portion thereof. Thus, the present invention is suitable for many applications, including experiments and screens addressing questions pertaining to innate and adaptive basic immunology, tumor immunology, oncology (e.g., as associated with immune cells), aging, drug testing, immunology associated with infection disease, autoimmune diseases, arthritis, and allergy, as well as for drug and vaccine development against these diseases. The present invention further provides methods and compositions for selecting cDNA clones for DNA arrays, wherein the clones are selected from diseased organisms, tissues, and/or cells.
In one embodiment, the clonal sequences necessary for the detection of changes in immune function in mice for inclusion on to the ImmunoChip have been deduced. In some embodiments, the ImmunoChip is a DNA microarray wherein the effects of disease progression and treatment protocols are monitored.
The arrays of the present invention are not limited to any particular means of construction. Indeed, the present invention contemplates arrays constructed by any suitable means. Many means of construction of arrays are known in the art (See e.g., the Detailed Description of the Invention).
The present invention contemplates that the arrays may be read by any suitable means, including manual and automated reading. In embodiments involving manual reading, typically reading is conducted visually xe2x80x9cby eyexe2x80x9d (e.g., the results are read by someone looking at and evaluating them). In alternative embodiments, instrumentation, such as microscopes or manual plate readers are used. Automated readers, for example scanners, are available commercially and are known to those in the art.
In one embodiment the present invention provides an array comprising the sequences of FIG. 3 (i.e., FIG. 3 shows the sequences of SEQ IN NOS: 1-29).
In one embodiment, the present invention provides a composition comprising an array of cDNA probes immobilized on a solid support, wherein the array comprises at least 100 probes and no more than 100,000 probes, which are approximately 100 to 2000 nucleotides in length occupying separate known sites in the array, and wherein at least a portion of the cDNA probes are selected from the sequences in FIG. 3. In another embodiment, the present invention contemplates that the probes are oligodeoxyribonuclotides. In yet another embodiment, the present invention provides oligodeoxynucleotide probes. In still another embodiment, the present invention provides an array that contains between 1,000 and 50,000 probes. In yet still another embodiment, the present invention provides an array that contains between 2,000 and 20,000 probes. In yet still another embodiment, the present invention provides an array that contains between 5,000 and 15,000 probes. In yet still another embodiment, the present invention provides a solid support that is selected from the group consisting of glass, plastic and metal.
The present invention provides compositions and devices comprising cDNA probe elements, probe sites or a plurality of probe molecules affixed to a solid microarray substrate. In one embodiment, the present invention comprises 10 probes selected from FIG. 3. In another embodiment, the present invention comprises 20 probes selected from FIG. 3. In most embodiments, probe elements containing different characteristic molecules are typically arranged in a two-dimensional array, for example, by microfluidic spotting techniques or by patterned photolithographic synthesis. In some embodiments, the present invention provides oligodeoxynulceotide probes, while in other embodiments, the probes are oligodeoxyribonucleotides In some embodiments, the present invention provides methods of selecting cDNA clones for DNA arrays, wherein the array has between 1,000 and 50,000 probes. In another embodiment, the present invention provides arrays having between 2,000 and 20,000 probes. In yet another embodiment, the present invention provides arrays having between 5,000 and 15,000 probes. In still further embodiments, the present invention provides a solid support that is selected from a group comprising glass, plastic and metal.
The present invention further provides methods of selecting DNA sequences for non-redundant microarrays, wherein the methods comprise: a) providing sequence database(s), b) screening the database(s) for DNA sequences specific for a species and/or a tissue found in the species, to generate a redundant sequence list; c) removing redundant sequences from the list to generate a non-redundant cluster list; d) categorizing selected sequences from non-redundant cluster list into at least one module list, and e) selecting the best representative clones based on the characteristics used to establish the parameters of the module list. In some embodiments of the present invention, the genes are screened from databases (for example, Unigen, GenBank, Locuslink, etc.) based on gene name or homolog name, the LL description, the PubMed indicated function, etc., (i.e., indicating the function or tissue desired). Redundant clones and duplicates are removed by importing the sequences into software programs designed for this purpose (for example, Panorama, ProVue, Huntington Beach, Calif.). The categorizing of the nonredundant clones is performed by putting clones with similar characteristics (e.g., whole genes, gene fragments, degrees of homology) into grouped called xe2x80x9cmodules.xe2x80x9d Selecting the best representative clones is performed by cluster score, blast score and/or physical parameters (e.g., size).
The present invention also provides methods selecting cDNA clones for DNA arrays, wherein the databases from which the clones are selected are from gene, bacterial, viral, phage, EST and image clone databases and literature sources, or any other suitable data source.